Monitoring of alpha-ketoglutarate in a fermentation process using expanded bed enzyme reactors.

A bienzyme flow injection system is presented for the monitoring of alpha-ketoglutarate produced in a fermentation process, using glutamate dehydrogenase (GDH) and glutamate oxidase (GlOx) immobilised in two serially connected expanded bed reactors. The use of expanded bed resulted in unhindered passage of the bacterial cells through the columns, and thereby the need of a separate filtering step (e.g. microdialysis) was avoided. In the first reactor, alpha-ketoglutarate was converted to L-glutamate by GDH in the presence of ammonia and NADH. In the following reactor, L-glutamate was converted by GlOx to alpha-ketoglutarate, ammonia and hydrogen peroxide, which was detected in an electrochemical flow-through cell at +650 mV vs. Pt/(0.1 M KCl). The detection limit of alpha-ketoglutarate in the coupled packed bed reactors was 1 microM (defined as 3 S/N), the linear range 0-100 microM, and the sensitivity 0.80 nA/microM (R(2) 0.99). In the coupled expanded bed reactors, the detection limit of alpha-ketoglutarate was 7 microM (defined as 3 S/N), the linear range and the sensitivity being 0-500 microM and 0.11 nA/microM (R(2) 1.00), respectively. The response time (defined as the time between peak rise and return to baseline) was 5 min for coupled packed beds (injection of supernatant), and 12 min for coupled expanded beds (injection of sample containing cellular and particulate matter). Several other parameters, such as reactor stability, flow rate dependency, bed expansion, glutamate interference, etc. were investigated and characterised. When analysing real samples from a fermentation broth, the same results were obtained independent of the nature of the reactor system (packed or expanded bed). The hereby described system can easily be automatised and controlled from a personal computer.     

Monitoring of α-ketoglutarate in a fermentation process using expanded bed enzyme reactors

A bienzyme flow injection system is presented for the monitoring of α-ketoglutarate produced in a fermentation process, using glutamate dehydrogenase (GDH) and glutamate oxidase (GlOx) immobilised in two serially connected expanded bed reactors. The use of expanded bed resulted in unhindered passage of the bacterial cells through the columns, and thereby the need of a separate filtering step (e.g. microdialysis) was avoided. In the first reactor, α-ketoglutarate was converted to -glutamate by GDH in the presence of ammonia and NADH. In the following reactor, -glutamate was converted by GlOx to α-ketoglutarate, ammonia and hydrogen peroxide, which was detected in an electrochemical flow-through cell at +650 mV vs. Pt/(0.1 M KCl). The detection limit of α-ketoglutarate in the coupled packed bed reactors was 1 μM (defined as 3 S/N), the linear range 0–100 μM, and the sensitivity 0.80 nA/μM (R² 0.99). In the coupled expanded bed reactors, the detection limit of α-ketoglutarate was 7 μM (defined as 3 S/N), the linear range and the sensitivity being 0–500 μM and 0.11 nA/μM (R² 1.00), respectively. The response time (defined as the time between peak rise and return to baseline) was 5 min for coupled packed beds (injection of supernatant), and 12 min for coupled expanded beds (injection of sample containing cellular and particulate matter). Several other parameters, such as reactor stability, flow rate dependency, bed expansion, glutamate interference, etc. were investigated and characterised. When analysing real samples from a fermentation broth, the same results were obtained independent of the nature of the reactor system (packed or expanded bed). The hereby described system can easily be automatised and controlled from a personal computer.     

An automated flow injection analysis system for on-line monitoring of glucose and L-lactate during lactic acid fermentation in a recycle bioreactor

The study concerns on-line sequential analysis of glucose and L-lactate during lactic acid fermentation using a flow injection analysis (FIA) system. Enzyme electrodes containing immobilized glucose oxidase and L-lactate oxidase were used with an amperometric detection system. A 12-bit data acquisition card with 16 analog input channels and 8 digital output channels was used. The software for data acquisition was developed using Visual C++, and was devised for sampling every hour for sequential analyses of lactate and glucose. The detection range was found to be 2–100 g l^–1 for glucose and 1–60 g l^–1 for L-lactate using the biosensors. This FIA system was used for monitoring glucose utilization and L-lactate production by immobilized cells of Lactobacillus casei subsp. rhamnosus during a lactic acid fermentation process in a recycle batch reactor. After 13 h of fermentation, complete sugar utilization and maximal L-lactate production was observed. A good agreement was observed between analysis data obtained using the biosensors and data from standard analyses of reducing sugar and L-lactate. The biosensors exhibited excellent stability during continuous operation for at least 45 days.     

Development of a D-alanine sensor for the monitoring of a fermentation using the improved selectivity by the combination of D-amino acid oxidase and pyruvate oxidase

A D-alanine (D-Ala) sensor for the monitoring of a fermentation process was developed using flow injection analysis (FIA). The FIA system consisted of a D-amino acid oxidase (D-AAOx) reactor, a Pyruvate oxidase (PyOx) electrode and a contrast electrode in the flow cell, and through the oxidation of D-amino acids in the D-AAOx reactor, pyruvic acid was formed only from D-Ala. The pyruvic acid was further oxidized with PyOx via the D-AAOx reaction. The amount of oxygen consumed in the PyOx reaction was proportional to the amount of D-Ala. It was possible to continuously repeat the assay up to 60 times at pH 6.8 and a flow rate of 0.18-ml min(-1). A linear relationship was obtained in the range of 0.1-1 mM D-Ala with a correlation coefficient of 0.987 and the detection limit was 0.05 mM. The relative standard deviation (R.S.D.) was 4.9% (n=5) for 0.5 mM D-Ala. The D-Ala content in some fish sauces was also determined using the proposed sensor system. The results obtained indicated a linear relationship between the amounts of D-Ala determined by the proposed sensor system and the conventional method. From the results, even if the substrate specificity of the enzyme (D-AAOx) was low, it was evident that the concentration of the original material (D-Ala) could be determined specifically when the first reaction product was changed by the second reaction (PyOx).     

Polyelectrolyte-coated ion exchangers for cell-resistant expanded bed adsorption

Adsorption chromatography in expanded beds is a widely used technology for direct capture of target proteins from fermentation broths. However, in many cases this method cannot be applied as a result of the strong tendency of cells or cell debris to interact with the adsorbent beads. To prevent contamination of the expanded bed with the biomass, STREAMLINE DEAE, anion exchanger designed for expanded bed adsorption, was modified with a layer of poly(acrylic acid) (PAA). The shielding layer of polyelectrolyte was attached to the surface of the matrix beads via electrostatic interactions. PAA with a high degree of polymerization was chosen to prevent diffusion of large polymer molecules into the pores of adsorbent. Thus, the shielding layer of PAA was adsorbed only at the mouth of the pores of STREAMLINE DEAE beads and only marginally decreased the binding capacity of the ion exchanger for bovine serum albumin, the model protein in this study. PAA-coated STREAMLINE DEAE practically did not interact with yeast cells, which otherwise bound strongly to the native adsorbent at neutral conditions. Cell-resistant PAA-coated anion exchanger was successfully used for isolation of BSA from the model protein mixture containing BSA, lysozyme (positively charged at applied conditions), and yeast cells. The layer of PAA was stable under mild elution conditions, and the modified adsorbent could be used in the repeated purification cycles.     

Partial purification properties of human epidermal growth factor from recombinant Escherichia coli by expanded bed adsorption

Human epidermal growth factor is a polypeptide hormone having many diverse biological functions. This paper first presents the recovery results of human epidermal growth factor (hEGF) immediately from the fermentation broth of recombinant Escherichia coli by using an expanded bed system (a couple of STREAMLINE25 and ÄKTA explorer 100). The influences of operational conditions such as linear flow rate, gradient length of NaCl concentration, pH and sample concentration on the purification performances of hEGF in expanded and packed bed modes with STREAMLINE DEAE resin were systematically evaluated. After optimization, the practical recovery procedure in the expanded bed mode was carried out on a scaled-up system under the conditions of linear flow rates of 183 cm/h (upward) and 37 cm/h (downward), sample volume of 300 ml and column bed height of 13.8 cm which yielded a primary product of hEGF from the cell-free supernatant containing hEGF after centrifugation at 4000 rev/min for 15 min. As a result, the hEGF concentration in the product was higher than 20% (w/v), the concentration factor was greater than 4.3 and the total yield was higher than 80%, respectively. At the same time, the results of hEGF recovery by using expanded bed adsorption (EBA), packed bed chromatography (PBC) and salting out were compared. The results show that the procedure of hEGF recovery in expanded bed adsorption has some advantages over the other two procedures, because of its higher concentration factor, recovery yield, productivity, hEGF concentration in the primary product and shorter duration of purification run.     

Binding assays in heterogeneous media using a flow injection system with an expanded micro-bed adsorption column

Competitive binding assays have been performed in flow injection systems. To further increase the versatility of the system, and to enable it to deal with samples containing particulate matter, the adsorption step was designed as an expanded bed column. Immunochemical quantification of human serum albumin was chosen as a model system to use for the development of the technology. A competitive ELISA was set up using peroxidase labelled HSA as competing ligand. The introduction of the expanded bed immunosorption column made the system tolerant to samples containing suspended particulate matter. The analytical outcome is very similar to that from the packed bed system even though more time is required for each assay cycle. The capability of the system was tested by addition of increasing amounts of yeast cells. The results clearly indicate that the system is suitable e.g. for process monitoring of fermentations.     

Offline glucose biomonitoring in yeast culture by polyamidoamine/cysteamine-modified gold electrodes

This article deals with the use of pyranose oxidase (PyOx) and glucose oxidase (GOx) enzymes in amperometric biosensor design and their application in monitoring fermentation processes with the combination of flow injection analysis (FIA). The amperometric studies were carried out at -0.7 V by following the oxygen consumption due to the enzymatic reactions for both batch and FIA modes. Optimization studies (enzyme amounts and pH) and analytical parameters such as linearity, repeatability, effect of interference, storage, and operational stabilities have been studied. Under optimized conditions, for the PyOx-based biosensor, linear graph was obtained from 0.025 to 0.5 mM glucose in phosphate buffer (50 mM) at pH 7.0 with the equation of y = 3.358x + 0.028 and R(2) = 0.998. Linearity was found to be 0.01-1.0 mM in citrate buffer (50 mM and pH 4.0) with the equation of y = 1.539x + 0.181 and R(2) = 0.992 for the GOx biosensor. Finally, these biosensor configurations were further evaluated in a conventional flow injection system. Results from batch experiments provide a guide to design sensitive, stable, and interference-free biosensors for FIA mode. Biosensor stability, dynamic range, and repeatability were also studied in FIA conditions, and the applicability for the determination of glucose in fermentation medium could be successfully demonstrated. The FIA-combined glucose biosensor was used for the offline monitoring of yeast fermentation. The obtained results correlated well with HPLC measurements.     

Use of a micro-expanded bed containing immobilised lysozyme for cell disruption in flow injection analysis

A method for cell disruption in Flow Injection Analysis (FIA) systems has been developed. The principle involves on-line cell disruption by means of immobilised lysozyme followed by an ultrasonic treatment. In order to avoid flow problems in the analytical system, the lysozyme was immobilised to Streamline^reg that was used in an expanded bed in the flow system. Samples of suspensions of Micrococcus lysodeikticus were treated and the success of the treatment was evaluated in terms of released protein and as a decrease in the optical density at 450 nm. The new technology offers a powerful tool in flow injection analyses for quantification of intracellular compounds. The concept of integration, i.e. combining cell disruption with handling of cell debris and assay procedure in one continuous flow process facilitates its use and increases the probability of reaching reproducible and reliable results.     

Analysis of micromolar concentrations of glucose by an interference free flow injection based biosensor

A fast, sensitive, interference-free, single enzyme single reagent glucose biosensor, operated in flow injection analysis (FIA) mode, was developed. The method used involved formation of colored complex of titanium sulfate reagent with the peroxide generated by glucose oxidase immobilized in a packed bed reactor. The color developed was detected spectrophotometrically in a flow cuvette. The system could measure down to 0.5 mg glucose l^–1 and the response was reproducible and linear in the range 1 mg l^–1 to 100 mg l^–1. The analysis time for a 500 l sample was 35 s and was free of interference from a number of substances tested. Analysis results using an off-line batch kit were observed to be in agreement with the developed system for determination of glucose in blood plasma samples.     

On-line monitoring of monoclonal antibody formation in high density perfusion culture using FIA

An automated flow injection system for on-line analysis of proteins in real fermentation fluids was developed by combining the principles of stopped-flow, merging zones flow injection analysis (FIA) with antigen-antibody reactions. IgG in the sample reacted with its corresponding antibody (a-IgG) in the reagent solution. Formation of insoluble immunocomplexes resulted in an increase of the turbidity which was determined photometrically. This system was used to monitor monoclonal antibody production in high cell density perfusion culture of hybridoma cells. Perfusion was performed with a newly developed static filtration unit equipped with hydrophilic microporous tubular membranes. Different sampling devices were tested to obtain a cell-free sample stream for on-line product anlysis of high molecular weight (e.g., monoclonal antibodies) and low molecular weight (e.g., glucose, lactate) medium components. In fermentation fluids a good correlation (coefficient: 0.996) between the FIA method and an ELISA test was demonstrated. In a high density perfusion cultivation process mAb formation was succesfully monitored on-line over a period of 400 h using a reliable sampling system. Glucose and lactate were measured over the same period of time using a commercially available automatic analyser based on immobilized enzyme technology.Abbreviations TIA Turbidimetric immunoassay - mAb Monoclonal Antibody     

On-line determination of intracellular beta-galactosidase activity in recombinant Escherichia coli using flow injection analysis (FIA).

A flow injection analysis (FIA) system was developed for the determination of cytoplasmic beta-galactosidase activity in recombinant Escherichia coli. The FIA system and its application for on-line monitoring of beta-galactosidase production during cultivation of recombinant E. coli in a 60-l airlift tower loop reactor is described. The results demonstrate that an FIA assay in conjunction with a cell disintegration step can be applied successfully for on-line monitoring of intracellular protein formation.     

Development of an expanded bed technique for an affinity purification of G6PDH from unclarified yeast cell homogenates

The use of an expanded bed of STREAMLINE Red H-7B for the purification of the intracellular glycolytic enzyme glucose 6-phosphate dehydrogenase (G6PDH) directly from untreated preparations of disrupted yeast cells has been investigated. Small-scale experiments, carried out in packed beds, have shown that the optimal pH for adsorption is 6.0 and have enabled optimization of elution conditions using a series of eluents. The dynamic capacity of the adsorbent for G6PDH was determined in a small expanded bed to be 28 units/mL. These results were used to develop a preparative scale separation of G6PDH in a STREAMLINE 50 expanded bed column. G6PDH was purified directly from an unclarified yeast homogenate in 99% yield with an average purification factor in the eluted fraction of 103. Cleaning-in-place (CIP) procedures using 0.5 M NaOH and 4M urea in 60% (v/v) ethanol have demonstrated that the adsorbent can be regenerated with no loss of adsorption capacity of alteration of bed expansion characteristics after many cycles of operation. (c) 1995 John Wiley & Sons, Inc.     

Characterisation of STREAMLINETM Phenyl

STREAMLINE Phenyl is a new hydrophobic interaction chromatography support designed for use in expanded bed adsorption. The phenyl groups are linked to STREAMLINE matrix via highly stable ether linkages. Within this development project the chemical and chromatographic stability as well as the breakthrough capacity for human IgG has been studied. The chemical stability was monitored as the carbon leakage from the matrix to the storage solution, pH 1–14 at 20 and 40 °C. The carbon content in the supernatant was determined with Total Organic Carbon (TOC) technique. In the chromatographic stability study STREAMLINE Phenyl was stored in eight different storage solutions under ambient conditions for 12 weeks and then tested in a chromatographic function test. The results show that the adsorbent is chemically stable and that the chromatographic properties are retained under the tested conditions. The breakthrough capacity study demonstrates the importance of the bed height for obtaining maximal dynamic capacity. Further, there is a good correlation between breakthrough data generated from packed bed and expanded bed runs.     

Ion exchange purification of G6PDH from unclarified yeast cell homogenates using expanded bed adsorption

The use of expanded beds of STREAMLINE ion exchange adsorbents for the direct extraction of an intracellular enzyme glucose-6-phosphate dehydrogenase (G6PDH) from unclarified yeast cell homogenates has been investigated. It has been demonstrated that such crude feedstocks can be applied to the bed without prior clarification steps. The purification of G6PDH from an unclarified yeast homogenate was chosen as a model system containing the typical features of a direct extraction technique. Optimal conditions for the purification were determined in small scale, packed bed experiments conducted with clarified homogenates. Results from these experiments were used to develop a preparative scale separation of G6PDH in a STREAMLINE 50 EBA apparatus. The use of an on-line rotameter for measuring and controlling the height of the expanded bed when operated in highly turbid feedstocks was demonstrated. STREAMLINE DEAE has been shown to be successful in achieving isolation of G6PDH from an unclarified homogenate with a purification factor of 12 and yield of 98% in a single step process. This ion exchange adsorbent is readily cleaned using simple cleaning-in-place procedures without affecting either adsorption or the bed expansion properties of the adsorbent after many cycles of operation. The ability of combining clarification, capture, and purification in a single step will greatly simplify downstream processing flowsheets and reduce the costs of protein purification. (c) 1996 John Wiley & Sons, Inc.     

Capture of proteins from mammalian cells in pilot scale using different STREAMLINE adsorbents

This presentation compares three different expanded bed matrices. STREAMLINE rProtein A, STREAMLINE SP-XL and STREAMLINE Chelating were monitored in respect to their ability to clarify the broth, to concentrate and to purify the distinct target protein. The capture of a mouse IgG₁ and a recombinant prothrombin (PT) was carried out in pilot scale using a 100-l bioreactor and STREAMLINE 100 and 200 columns, respectively. The robustness of the process was also estimated monitoring the expansion behaviour and the cell and debris concentrations during the load and in the eluat. In all cases the capture of the target proteins was comparable to conventional chromatographic systems. The purification success was mainly dependent on the selectivity of the ligand used. The affinity process resulted in a highly purified product. The ion exchanger and chelating material mainly concentrated the product. In all three cases 100 l of cell broth were successfully processed in one run. The robustness of the ion exchanger process was poor, because of strong cell matrix interaction. However, for the chelating and especially for the affinity matrix a highly reproducible process was obtained.     

A novel continuous centrifugal bioreactor for high-density cultivation of mammalian and microbial cells

A continuous centrifugal bioreactor (CCBR), developed to study the growth and productivity of dense suspensions cultures, has been applied to both fermentation and mammalian cell cultivation processes. With this approach, high-density nonflocculent cultures are maintained in a tapered fluidized bed by balancing the drag forces on the cells due to following substrate with the centrifugal forces. The Sysyem was first used to produce ethanol by fermentation with Saccharomyces cerevisiae; then with H21A1 mouse hybridoma cells secreting monoclonal antibody (MoAb), lgM. Results of this research show the feasibility of using the CCBR for both production of secreted products and as a research tool for studying cell metabolism and production kinetics. Media recycle may be used to modify the behavior of the system form a plug flow apparatus to a continuous stirred reactor (CSTR).     

Cell separation by expanded bed adsorption: use of ion exchange chromatography for the separation of <Emphasis Type="Italic">E. coli</Emphasis> and <Emphasis Type="Italic">S. cerevisiae</Emphasis>

In a wide variety of biotechnological and medical applications it is necessary to separate different cell populations from one another. A promising approach to cell separations is demonstrated to be the adoption of chromatographic techniques conducted in expanded beds. The high voidage between the adsorbent beads in an expanded bed allows for the efficient capture of particulate entities such as cells together with washing and subsequent elution without entrapment and loss. In addition, the combination of a gentle hydrodynamic environment, a high surface area and low mixing within the expanded bed make this technique highly favourable. A model system for the separation of two types of microbial cells using STREAMLINE DEAE adsorbent in expanded bed procedures has been investigated. The use of a less selective ligand such as an ion exchange group, which is often characterised by gentle elution procedures, has been investigated as an alternative to affinity ligands whose strong binding characteristics can result in harsh elution procedures with consequent loss of yield and cell viability. Expanded bed experiments have demonstrated selective and high capacity capture of cells from feedstocks containing either a single type of cell or as a mixture of cells of Saccharomyces cerevisiae and Eschericia coli. The capture, washing and elution phases of the separation have been studied with respect to capacity, selectivity and yield of released cells. In these procedures, separation of cell types is achieved by the presence of multiple equilibrium stages within the expanded bed. The results show the potential for carrying out cell separations in expanded beds as an alternative to immunomagnetic cell separations. The combination of these recently developed technologies promises to be a powerful, but economic technique for cell separations involving simple equipment that can readily be scaled up.     

Development and scale up of a capture step (expanded bed chromatography) for a fusion protein expressed intracellularly in Escherichia coli

A capture step was developed using the expanded bed adsorption technology to separate a protein of interest on a cation exchanger from a crude Escherichia coli homogenate. This method was developed in bench-top scale using a STREAMLINE 25 column (Amersham Pharmacia Biotech, Sweden) and STREAMLINE SP. The development was based on earlier experiments performed in a packed bed column (SP-Sepharose FF) to investigate the conditions for sample application, wash and elution. The packed bed method was transformed into an expanded bed method by slightly modifying the wash procedure and cleaning in place (CIP). This method was then scaled-up to pilot scale and used for production of the fusion protein according to cGMP.The yield over the step in pilot scale was 70-85% compared with only 30-50% in small scale. Pressure build-up, attachment of biomass to the adsorbent and collapses of the expanded bed were phenomena seen in small scale but not in pilot scale. The scale-up of the step significantly improved the performance of the step.     

Determination of l-glutamate in various commercial soy sauce products using flow injection analysis with a modified electrode

A flow injection analysis (FIA) system with a modified electrode has been developed and optimized for determination of l-glutamate using l-glutamate oxidase (GLOD) (EC 1.4.3.11). GLOD was immobilized on controlled-pore glass using glutaraldehyde. The optimal potential applied on the working electrode was +700mV against a platinum (Pt) reference electrode. The optimal pH and flow rate of the carrier buffer were 7.4 and 1.5ml/min, respectively. A modified electrode was integrated into the FIA system in order to eliminate electroactive interference and it was used to determine l-glutamate in 39 samples of Thai commercial soy sauce products. The results obtained were compared with those obtained from enzymatic assay using glutamate dehydrogenase and those from a chromatographic assay using an amino acid analyser. Good correlations were observed amongst these methods. The results indicated that use of an FIA system with a modified electrode was able to eliminate electroactive interference and was applicable to the determination of l-glutamate in food samples. The modified FIA was faster and simpler than the more common methods of enzymatic and chromatographic analysis.